Slit2/Robo4信号通路在输血相关急性肺损伤体外模型中的表达及作用

doi:10.3877/cma.j.issn.1674-6880.2018.03.001

目的

探讨Slit2/Robo4信号通路在输血相关急性肺损伤（TRALI）体外模型中的表达及作用。

方法

采用两次打击多形核中性粒细胞（PMNs）介导的人肺微血管内皮细胞（HMVECs）损伤模型作为TRALI体外模型，培养0、0.5、1、2、4、6 h后，采用Western-blotting法检测Robo4和血管内皮细胞钙粘连蛋白（VE-cadherin）表达，反转录酶PCR（RT-PCR）检测Robo4和Slit2信使RNA（mRNA）表达，体外内皮细胞通透性实验测定HMVECs通透性。加入不同浓度Slit2-N（0、0.5、1、4、10、20 μg/L）与HMVECs一起温孵24 h后，检测HMVECs完整性和通透性。

结果

在TRALI体外模型中，Robo4和Slit2 mRNA的表达在各时间点的比较，差异均有统计学意义（F=12.880、11.060，P均< 0.001）；两者在2 h（0.72 ± 0.04、0.78 ± 0.05）、4 h（0.49 ± 0.04、0.49 ± 0.06）和6 h（0.34 ± 0.03、0.43 ± 0.11）均显著低于0 h（1.29 ± 0.06、1.40 ± 0.09），差异均有统计学意义（P均< 0.05）。Robo4蛋白表达在各时间点的比较，差异有统计学意义（F=11.560，P < 0.001）；其在2、4和6 h（0.99 ± 0.04、0.66 ± 0.03、0.45 ± 0.04）均显著低于0 h（1.44 ± 0.04），差异均有统计学意义（P均< 0.05）。同时发现，VE-cadherin蛋白表达在各时间点的比较，差异也有统计学意义（F=9.667，P < 0.001）；与0 h（1.46 ± 0.09）比较，VE-cadherin的表达在2、4和6 h（0.91 ± 0.08、0.78 ± 0.05、0.50 ± 0.04）也均有减少，差异均有统计学意义（P均< 0.05）。体外内皮细胞渗透性试验提示，HMVECs通透性在各时间点的比较，差异有统计学意义（F=21.940，P < 0.001）；与0 h（1.42 ± 0.16）比较，HMVECs通透性在2、4、6 h（4.00 ± 0.35、5.70 ± 1.71、10.02 ± 2.24）均有增加，差异均有统计学意义（P均< 0.05）。加入外源性Slit2-N后，VE-cadherin蛋白表达的比较，差异有统计学意义（F=13.220，P < 0.001）；与0 μg/L Slit2-N组（0.41 ± 0.08）相比，VE-cadherin在4、10、20 μg/L Slit2-N组表达（1.19 ± 0.35、1.49 ± 0.13、2.12 ± 0.21）均有增加，差异均有统计学意义（P均< 0.05）。体外内皮细胞渗透性实验提示，HMVECs通透性比较，差异有统计学意义（F=19.430，P < 0.001）；与0 μg/L Slit2-N组（10.0 ± 2.2）相比，HMVECs通透性在4、10、20 μg/L Slit2-N组（4.2 ± 1.1、2.1 ± 0.7、1.8 ± 0.8）均有降低，差异均有统计学意义（P均< 0.05）。

结论

Slit2/Robo4信号通路可能参与TRALI的病理生理过程；使用外源性Slit2-N能调节TRALI体外模型中HMVECs的完整性和通透性，为治疗TRALI提供新的思路。

关键词: 输血相关急性肺损伤, Slit2/Robo4, 血管内皮细胞钙粘连蛋白, 人肺微血管内皮细胞

Objective

To investigate the expression and effect of Slit2/Robo4 signaling pathway in vitro model of transfusion related acute lung injury (TRALI).

Methods

A two-hit model of polymorphonuclear neutrophils (PMNs)-mediated human pulmonary microvascular endothelial cells (HMVECs) damage was used as TRALI in vitro model. After the HMVECs were incubated for 0, 0.5, 1, 2, 4, and 6 h, the protein expressions of Robo4 and vascular endothelial cadherin (VE-cadherin) were detected by Western-blotting, and the mRNA expressions of Robo4 and Slit2 were analyzed by reverse transcription PCR (RT-PCR). The permeability of HMVECs was detected by in vitro experiment of endothelial cell permeability. Then the HMVECs were incubated with 0, 0.5, 1, 4, 10, and 20 μg/L Slit2-N for 24 h. The integrity and permeability of HMVECs were detected.

Results

In vitro model of TRALI, the mRNA expressions of Robo4 and Slit2 both showed significant differences at different time points (F=12.880, 11.060; both P < 0.001); they were much lower at 2 h (0.72 ± 0.04, 0.78 ± 0.05), 4 h (0.49 ± 0.04, 0.49 ± 0.06), and 6 h (0.34 ± 0.03, 0.43 ± 0.11) than those at 0 h (1.29 ± 0.06, 1.40 ± 0.09; all P < 0.05). The protein expression of Robo4 showed significant difference at each time point (F=11.560, P < 0.001); it decreased at 2, 4, 6 h (0.99 ± 0.04, 0.66 ± 0.03, 0.45 ± 0.04) in comparing with 0 h (1.44 ± 0.04, all P < 0.05). In addition, the protein expression of VE-cadherin at each time point was significantly different (F=9.667, P < 0.001); its expression decreased significantly at 2, 4, 6 h (0.91 ± 0.08, 0.78 ± 0.05, 0.50 ± 0.04) in comparing with 0 h (1.46 ± 0.09, all P < 0.05). The vitro experiment of endothelial cell permeability revealed that the permeability of HMVECs was significant different at each time point (F=21.940, P < 0.001); as comparing with 0 h (1.42 ± 0.16), the permeability of HMVECs was significantly higher at 2, 4, 6 h (4.00 ± 0.35, 5.70 ± 1.71, 10.02 ± 2.24; all P < 0.05). After adding the exogenous Slit2-N, the expression of VE-cadherin protein was statistically significantly different (F=13.220, P < 0.001); it increased in the 4, 10, 20 μg/L Slit2-N groups (1.19 ± 0.35, 1.49 ± 0.13, 2.12 ± 0.21) as comparing with the 0 μg/L Slit2-N group (0.41 ± 0.08, all P < 0.05). The vitro experiment of endothelial cell permeability revealed that the permeability of HMVECs was significant different (F=19.430, P < 0.001); the permeability of HMVECs was significantly lower in the 4, 10, 20 μg/L Slit2-N groups (4.2 ± 1.1, 2.1 ± 0.7, 1.8 ± 0.8) as comparing with the 0 μg/L Slit2-N group (10.0 ± 2.2, all P < 0.05).

Conclusion

The Slit2/Robo4 signaling pathway may be involved in the pathophysiological process of TRALI. Exogenous Slit2-N may modulate the integrity and permeability of HMVECs in vitro TRALI model, and become a promising candidate for developing novel therapies against TRALI.

Key words: Transfusion related acute lung injury, Slit2/Robo4, VE-cadherin, Human pulmonary microvascular endothelial cell

表1 不同时间点Robo4、Slit2在TRALI体外模型中的表达（±s）

各时间点	细胞浓度（个/mL）	Robo4蛋白	Robo4 mRNA	Slit2 mRNA
0 h	1 × 10⁶	1.44 ± 0.04	1.29 ± 0.06	1.40 ± 0.09
0.5 h	1 × 10⁶	1.30 ± 0.04	1.26 ± 0.08	1.34 ± 0.05
1 h	1 × 10⁶	1.24 ± 0.07	0.94 ± 0.09	1.15 ± 0.19
2 h	1 × 10⁶	0.99 ± 0.04^a	0.72 ± 0.04^a	0.78 ± 0.05^a
4 h	1 × 10⁶	0.66 ± 0.03^a	0.49 ± 0.04^a	0.49 ± 0.06^a
6 h	1 × 10⁶	0.45 ± 0.04^a	0.34 ± 0.03^a	0.43 ± 0.11^a
F值	?	11.560	12.880	11.060
P值	?	<0.001	<0.001	<0.001

表1 不同时间点Robo4、Slit2在TRALI体外模型中的表达（±s）

各时间点	细胞浓度（个/mL）	Robo4蛋白	Robo4 mRNA	Slit2 mRNA
0 h	1 × 10⁶	1.44 ± 0.04	1.29 ± 0.06	1.40 ± 0.09
0.5 h	1 × 10⁶	1.30 ± 0.04	1.26 ± 0.08	1.34 ± 0.05
1 h	1 × 10⁶	1.24 ± 0.07	0.94 ± 0.09	1.15 ± 0.19
2 h	1 × 10⁶	0.99 ± 0.04^a	0.72 ± 0.04^a	0.78 ± 0.05^a
4 h	1 × 10⁶	0.66 ± 0.03^a	0.49 ± 0.04^a	0.49 ± 0.06^a
6 h	1 × 10⁶	0.45 ± 0.04^a	0.34 ± 0.03^a	0.43 ± 0.11^a
F值	?	11.560	12.880	11.060
P值	?	<0.001	<0.001	<0.001

表2 TRALI体外模型中VE-cadherin表达及HMVECs通透性在不同时间点的比较（±s）

各时间点	细胞浓度（个/mL）	VE-cadherin蛋白	HMVECs通透性
0h	1 × 10⁶	1.46 ± 0.09	1.42 ± 0.16
0.5 h	1 × 10⁶	1.31 ± 0.08	1.61 ± 0.49
1h	1 × 10⁶	1.13 ± 0.05	2.06 ± 0.21
2h	1 × 10⁶	0.91 ± 0.08^a	4.00 ± 0.35^a
4h	1 × 10⁶	0.78 ± 0.05^a	5.70 ± 1.71^a
6h	1 × 10⁶	0.50 ± 0.04^a	10.02 ± 2.24^a
F值	?	9.667	21.940
P值	?	<0.001	<0.001

表2 TRALI体外模型中VE-cadherin表达及HMVECs通透性在不同时间点的比较（±s）

各时间点	细胞浓度（个/mL）	VE-cadherin蛋白	HMVECs通透性
0h	1 × 10⁶	1.46 ± 0.09	1.42 ± 0.16
0.5 h	1 × 10⁶	1.31 ± 0.08	1.61 ± 0.49
1h	1 × 10⁶	1.13 ± 0.05	2.06 ± 0.21
2h	1 × 10⁶	0.91 ± 0.08^a	4.00 ± 0.35^a
4h	1 × 10⁶	0.78 ± 0.05^a	5.70 ± 1.71^a
6h	1 × 10⁶	0.50 ± 0.04^a	10.02 ± 2.24^a
F值	?	9.667	21.940
P值	?	<0.001	<0.001

表3 在不同浓度Slit2-N作用下TRALI体外模型中VE-cadherin表达及HMVECs通透性的比较（±s）

Slit2-N浓度（μg/L）	细胞浓度（个/mL）	VE-cadherin蛋白	HMVECs通透性
0	1 × 10⁶	0.41 ± 0.08	10.0 ± 2.2
0.5	1 × 10⁶	0.61 ± 0.06	10.1 ± 1.3
1	1 × 10⁶	0.68 ± 0.15	9.0 ± 1.4
4	1 × 10⁶	1.19 ± 0.35^a	4.2 ± 1.1^a
10	1 × 10⁶	1.49 ± 0.13^a	2.1 ± 0.7^a
20	1 × 10⁶	2.12 ± 0.21^a	1.8 ± 0.8^a
F值	?	13.220	19.430
P值	?	<0.001	<0.001

表3 在不同浓度Slit2-N作用下TRALI体外模型中VE-cadherin表达及HMVECs通透性的比较（±s）

Slit2-N浓度（μg/L）	细胞浓度（个/mL）	VE-cadherin蛋白	HMVECs通透性
0	1 × 10⁶	0.41 ± 0.08	10.0 ± 2.2
0.5	1 × 10⁶	0.61 ± 0.06	10.1 ± 1.3
1	1 × 10⁶	0.68 ± 0.15	9.0 ± 1.4
4	1 × 10⁶	1.19 ± 0.35^a	4.2 ± 1.1^a
10	1 × 10⁶	1.49 ± 0.13^a	2.1 ± 0.7^a
20	1 × 10⁶	2.12 ± 0.21^a	1.8 ± 0.8^a
F值	?	13.220	19.430
P值	?	<0.001	<0.001

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Expression and effect of Slit2/Robo4 signaling pathway in vitro model of transfusion related acute lung injury

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Expression and effect of Slit2/Robo4 signaling pathway in vitro model of transfusion related acute lung injury